RhoA/ROCK I signalling downstream of the P2Y13 ADP-receptor controls HDL endocytosis in human hepatocytes.
Cell surface receptors for high-density lipoprotein (HDL) on hepatocytes are major partners in the regulation of cholesterol homeostasis. We have previously demonstrated on human hepatocytes that apolipoprotein A-I binding to an ectopic F(1)-ATPase stimulates the production of extracellular ADP that activates a P2Y(13)-mediated high-density lipoprotein (HDL) endocytosis pathway. However, P2Y(13)-dependent signalling pathway has never been described yet. The current study demonstrates a major role of cytoskeleton reorganization in F(1)-ATPase/P2Y(13)-dependent HDL endocytosis under the control of the small GTPase RhoA and its effector ROCK I. Indeed human hepatocytes (HepG(2) cells) stimulated by ADP or AR-C69931MX (both P2Y(13) agonists) showed a high specific activation of RhoA; in addition, inhibition of Rho proteins by C3 exoenzyme impairs HDL endocytosis whereas a constitutively active form of RhoA stimulates HDL endocytosis at the same level as under F(1)-ATPase/P2Y(13) activation. Pharmacological inhibition of ROCK activity decreased HDL endocytosis following stimulation by apoA-I (F(1)-ATPase ligand), ADP or AR-C69931MX and specific siRNA ROCK I extinction prevented the stimulation of HDL endocytosis without effect of ROCK II extinction. The functional involvement of ROCK I downstream F(1)-ATPase/P2Y(13) was confirmed by the strong enrichment of the membrane fraction in ROCK I and by the requirement of actin polymerization in hepatocyte HDL endocytosis. These results allow the identification of the molecular events downstream P2Y(13) receptor activation for a better understanding of hepatocyte HDL endocytosis, the latest step in reverse cholesterol transport.
Malaval
Camille
C
INSERM, U563, Toulouse, F-31000, France.
Laffargue
Muriel
M
Barbaras
Ronald
R
Rolland
Corinne
C
Peres
Christine
C
Champagne
Eric
E
Perret
Bertrand
B
Tercé
François
F
Collet
Xavier
X
Martinez
Laurent O
LO
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000256:Adenoviridae / Q000235:genetics; D016632:Apolipoprotein A-I / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D004705:Endocytosis; D022781:Hepatocytes / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D008075:Lipoproteins, HDL / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism; D058910:Purinergic P2 Receptor Agonists; D034741:RNA, Small Interfering / Q000235:genetics; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D015398:Signal Transduction; D054460:rho-Associated Kinases / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; C440036:P2RY13 protein, human; D058910:Purinergic P2 Receptor Agonists; D034741:RNA, Small Interfering; D018048:Receptors, Purinergic P2; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; C117446:cangrelor; D054460:rho-Associated Kinases; D006180:Proton-Translocating ATPases; D020742:rhoA GTP-Binding Protein